Download Disruption of the long-range GPIIIa Cys5

From www.bloodjournal.org by guest on August 11, 2017. For personal use only.
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Disruption of the long-range GPIIIa Cys5-Cys435 disulfide bond results in the
production of constitutively active GPIIb-IIIa (␣IIb␤3) integrin complexes
Qi-Hong Sun, Chao-Yan Liu, Ronggang Wang, Cathy Paddock, and Peter J. Newman
The major platelet integrin ␣IIb␤3, also
known as the platelet glycoprotein (GP)
IIb-IIIa complex, mediates platelet aggregation by serving as the receptor for
fibrinogen and von Willebrand factor. In
addition to its physiologic role, GPIIb-IIIa
also bears a number of clinically important alloantigenic determinants. Previous
studies have shown that disruption of the
long-range Cys5-Cys435 disulfide bond of
the ␤3 subunit results in the production of
isoforms that bind some, but not all,
anti-PlA1 alloantibodies, suggesting that
mutations in this so-called long-range
disulfide bond can alter the conformation
of GPIIIa. The purpose of this study was
to examine the effects of either the
Cys5Ala or Cys435Ala substitution of
GPIIIa on the adhesive properties of the
GPIIb-IIIa complex. We found that both
Ala5GPIIIa and Ala435GPIIIa were capable of associating with GPIIb and were
expressed normally on the cell surface
when cotransfected into Chinese hamster ovary (CHO) cells. CHO cells expressing GPIIb-Ala5GPIIIa or GPIIb-Ala435IIIa
bound well-characterized, conformationally sensitive ligand-induced binding site
(LIBS) antibodies, and were capable of
constitutively binding the fibrinogenmimetic monoclonal antibodies Pl-55 and
PAC-1, as well as soluble fibrinogen. Both
GPIIb-Ala5IIIa– and GPIIb-Ala435IIIa–
transfected CHO cells also bound more
avidly to immobilized fibrinogen and were
capable of mediating the tyrosine phosphorylation of pp125FAK on cell adhesion.
These data are consistent with the notion
that these regions of GPIIIa participate in
the conformational change associated
with receptor activation. Additionally,
these studies may provide a molecular
explanation for the previously reported
ability of mild reducing agents to activate
the GPIIb-IIIa complex and promote platelet aggregation. (Blood. 2002;100:
2094-2101)
© 2002 by The American Society of Hematology
Introduction
Integrins, one of several gene families that encode cell surface
adhesive receptors capable of mediating cell-cell and cellextracellular matrix (ECM) interactions,1 are heterodimers consisting of a 120- to 180-kDa ␣ subunits noncovalently associated with
a 90- to 110-kDa ␤ subunits. To date, 19 ␣ and 8 ␤ subunits have
been characterized, and 24 members of this heterodimer family
have been identified.2,3 Integrins mediate both adhesion and
bidirectional transmembrane signaling. The binding of these integrins to soluble macromolecules is tightly regulated by integrin
activation or inside-out signaling, also known as affinity modulation, which involves structural changes intrinsic to the heterodimer,
and avidity modulation due to lateral diffusion and clustering of
heterodimers into oligomers.4,5 In addition, ligand-induced binding
site (LIBS) antibodies, which preferentially bind the ligandoccupied form of receptor, can lock the integrin into a higheraffinity state without the need for signals from inside the cell.6,7
Following receptor occupancy, information is transduced across
the plasma membrane in a process termed outside-in signaling.
These signals include elevation of intracellular pH and calcium,
inositol lipid synthesis, and the tyrosine phosphorylation of a wide
range of proteins, including focal adhesion kinase (FAK), Src, and
adaptor proteins such as Shc and p130 CAS. These signaling
events, in turn, trigger a range of downstream signals, including
activation of the Ras/mitogen-activated protein (MAP) kinase
pathway.8
The platelet glycoprotein (GP) GPIIb-IIIa complex (integrin
␣IIb␤3) is a well-characterized example of dynamic regulation of
integrin function. GPIIb-IIIa is inactive in resting platelets, and
does not bind soluble fibrinogen or von Willebrand factor (VWF).
Following cellular stimulation, however, GPIIb-IIIa undergoes a
rapid conformational change that results in the exposure of one or
more ligand contact sites and binds its ligands with high affinity.
The altered ligand-binding affinity is due to a change in the
conformation of the external domain of the receptor, which allows
better access for macromolecular ligands to the ligand-binding
domains, which are localized to the large globular head of the
receptor. Ligand binding to GPIIb-IIIa complex further modifies
the conformation of this integrin,9 leading to postreceptor occupancy events such as clustering of GPIIb-IIIa, tyrosine phosphorylation, and cytoskeleton rearrangement.10,11
GPIIIa (the integrin ␤3 subunit) consists of 762 amino acids,
and like other integrin ␤ subunits, is comprised of a large
extracellular domain, a single membrane-spanning region, and a
47-amino acid cytoplasmic tail.12 Recent crystallographic studies
From the Blood Research Institute, Blood Center of Southeastern Wisconsin,
and the Cardiovascular Center, Departments of Pharmacology and Cellular
Biology, Medical College of Wisconsin, Milwaukee.
Q-H.S. and C-Y.L. contributed equally to this work.
Submitted February 28, 2002; accepted May 6, 2002. Prepublished online as
Blood First Edition Paper, May 24, 2002; DOI 10.1182/blood-2002-02-0418.
Supported by Grant-in-Aid Award 0050581N (Q-H.S.) from the American Heart
Association, and by Program Project grant P01 HL44612-12 (P.J.N.) from the
National Institutes of Health.
2094
Reprints: Qi-Hong Sun, Blood Research Institute, The Blood Center of
Southeastern Wisconsin, 8727 Watertown Plank Rd, PO Box 2178, Milwaukee,
WI 53233; e-mail: [email protected].
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2002 by The American Society of Hematology
BLOOD, 15 SEPTEMBER 2002 䡠 VOLUME 100, NUMBER 6
From www.bloodjournal.org by guest on August 11, 2017. For personal use only.
BLOOD, 15 SEPTEMBER 2002 䡠 VOLUME 100, NUMBER 6
of ␣v␤3 confirms earlier visualization13 of the overall shape of
integrins as containing a large ligand-binding “head” on top of ␣v
and ␤3 subunit “legs.”14 A ␤A ligand-binding domain (residues
109-352) looping out from a unique immunoglobulin (Ig)-like
“hybrid” domain (residues 55-108 and 353-434) in ␤3 forms the
␣v␤3 head, which is in close apposition with a 7-bladed ␤ propeller
in the ␣v NH2-terminal segment. The ␤3 subunit leg includes a PSI
(plexins, semaphorins, and integrins) domain (residues 1-54), 4
cysteine-rich epidermal growth factor (EGF)–like repeats (residues
453-605), and a ␤-terminal domain (␤-TD, residues 606-690). A
distinguishing feature of GPIIIa, and presumably other integrin ␤
subunits, is a proposed “long-range” disulfide bond formed by the
pairing of Cys5 with Cys435,15 which, if present, would connect the
PSI domain to a small, flexible region termed “linker 2,” which is
immediately amino-terminal the first EGF repeat (residues 435452). Unfortunately, the disulfide bond between Cys5 and Cys435
could not be directly visualized in the ␤3 crystal structure.14
Although all of cysteines within GPIIIa have been thought to form
disulfide bonds that stabilize the overall 3-dimensional structure,
recent studies suggest that EGF repeats 2 and 4 may contain
unpaired cysteines, which exhibit the properties of a redox site
involved in integrin activation.16 In concert with these findings,
GPIIIa has also been reported to contain endogenous thiol isomerase activity, predicted from the presence of the tetrapeptide motif,
CXXC, in each of GPIIIa EGF repeats.17 Disulfide exchange has
been demonstrated to take place during integrin-mediated platelet
adhesion and surface thiol isomerase has been implicated in this
process.18
A number of gain-of-function GPIIIa mutations have been
experimentally induced and studied in recombinant GPIIb-IIIa–
transfected cells. Bajt et al demonstrated that replacement of
residues 129 to 133 within the ligand-binding site of GPIIIa with
the corresponding sequence from the integrin ␤1 subunit resulted in
increased fibrinogen binding.19 A recombinant soluble form of
GPIIb-IIIa has been found to assume an active, ligand-binding
conformation and is recognized by GPIIb-IIIa–specific monoclonal, allo-, auto-, and drug-dependent platelet antibodies.20 Truncation of the EGF repeats of GPIIIa leads to GPIIb-IIIa exhibiting
enhanced binding capacities for fibrinogen.21 Recently, a Thr562Asn
mutation located within the ␤3 EGF repeat region was identified
during screening Chinese hamster ovary (CHO) cell transfectants
for high-affinity variants of GPIIb-IIIa and ␣v␤3,22 and Cys598Tyr
mutation in GPIIIa has been reported to induce spontaneous
binding of the ligand mimetic monoclonal antibody PAC-1.23
Deletion or mutation in conserved sequences within ␤3 membraneproximal cytoplasmic domain also has been found to result in an
activated complex locked in a high-affinity state.24 In this report,
we provide evidence that disruption of the long-range GPIIIa
Cys5-Cys435 disulfide bond by substituting Ala for Cys at position 5
or 435 of GPIIIa results in the production of constitutively active
GPIIb-IIIa integrin complexes. These data also provide functional
confirmation for the presence of a long-range disulfide bond
between Cys5-Cys435, and support the notion that this loop participates in the conformational changes associated with receptor
activation.
Materials and methods
Materials
The GPIIb-IIIa complex-specific monoclonal antibody, AP2,25 and GPIIIaspecific monoclonal antibodies, AP326 and AP5,9 were produced at the
CONSTITUTIVELY ACTIVE GPIIb-IIIa COMPLEXES
2095
Blood Research Institute Hybridoma Core Laboratory and purified by
affinity chromatography. The LIBS antibody, D3,27 was provided by Dr
Lisa Jennings (University of Tennessee, Memphis), 7G2 by Dr Eric Brown
(Washington University School of Medicine, St Louis, MO), and 7C7 by Dr
Nathalie Valentin (Centre Regional de Transfusion Sanguine, Nantes,
France), and CRC54 by Dr Alexy Mazurov (Institute of Experimental
Cardiology, Cardiology Research Center, Moscow, Russian Federation).28
Fibrinogen mimetic anti–GPIIb-IIIa antibodies PAC-129,30 (murine IgM)
and Pl-5531 were generously provided by Drs Sandy Shattil (Scripps
Research Institute, La Jolla, CA), and Beat Steiner (Hoffmann-La Roche,
Basel, Switzerland), respectively. RGDW and RGEW peptides were
synthesized using a Model 9050 Pepsynthesizer (Millipore, Bedford, MA)
with Fmoc chemistry at the Blood Research Institute Peptide
Core Laboratory.
Cells and cell lines
Nucleotide substitutions were introduced into full-length GPIIIa cDNA in
mammalian expression vector pcDNA3 and the constructs were analyzed
by automated sequencing (Applied Biosystems, Foster City, CA) using
methods previously described by Valentin et al.32 CHO cells were
cotransfected with GPIIb (in mammalian expression vector EMC-3) and
different GPIIIa (wild-type [WT] GPIIIa, Ala5GPIIIa, or Ala435GPIIIa in
pcDNA3) using the standard calcium phosphate method.33 After 48 hours,
transfected CHO cells were selected in ␣-minimum essential media (MEM)
without ribonucleotides or deoxyribonucleotides containing 600 ␮g/mL
G418 (geneticin, Gibco, Gaithersburg, MD) for about 2 weeks. To obtain
stable cell lines with high and comparable level of expression, the
transfected CHO cells were sorted in a FACStar (Becton Dickinson, San
Jose, CA) using AP3, cloned by limited dilutions, and CHO cells expressing
GPIIb and different GPIIIa were maintained in selection media containing 5
nM methotrexate.
Western blot analysis of expressed GPIIIa
Stable CHO cell lines expressing WT GPIIb-IIIa, GPIIb-Ala5IIIa, or
GPIIb-Ala435IIIa were harvested, washed, and solubilized in lysis buffer
either containing 50 mM Tris (tris(hydroxymethyl)aminomethane), 2%
sodium dodecyl sulfate (SDS), 10 mM N-ethylmaleimide, 2 mM phenylmethylsulfonyl fluoride (PMSF), and 100 ␮g/mL leupeptin (for nonreducing
condition), or containing 50 mM Tris, 2% SDS, and 5% ␤-mercaptoethanol
(for reducing condition) at 4°C. After centrifugation at 15 000g for 30
minutes, soluble lysates were electrophoresed on 7% SDS-polyacrylamide
gel electrophoresis (PAGE), and transferred to polyvinylidene difluoride
(PVDF) membranes (Millipore). The membranes were blocked with 3%
bovine serum albumin (BSA) and incubated with well-characterized rabbit
polyclonal antibodies specific for GPIIIa.34 After washing, the membrane
was incubated with goat-anti–rabbit IgG conjugated with horseradish
peroxidase, followed by chemiluminescence detection according to the
manufacturer’s instructions (Amersham Life Science, Piscataway, NJ).
Immunoprecipitation of GPIIb-IIIa complex from lysates
of the transfected CHO cells
Nontransfected CHO cells and transfected CHO cells expressing WT
GPIIb-IIIa, GPIIb-Ala5IIIa, or GPIIb-Ala435IIIa were surface-labeled with
5 mM NHS-LC-biotin (Pierce, Rockford, IL) in phosphate-buffered saline
(PBS) for 30 minutes at 22°C, and solubilized in lysis buffer (20 mM Tris,
100 mM NaCl, 1% Triton X-100, 2 mM PMSF, and 100 ␮g/mL leupeptin)
for 30 minutes on ice.35 The supernatant was obtained by centrifugation at
15 000g for 30 minutes at 4°C. Aliquots of biotin-labeled cell lysates were
precleared, and incubated overnight at 4°C with monoclonal antibodies
(mAbs) specific for GPIIIa subunit, the GPIIb-IIIa complex, or normal
mouse IgG (NMIgG). Rabbit anti–mouse IgG was added, and the immune
complexes were recovered with protein A Sepharose beads (Pharmacia
Biotech, Uppsala, Sweden). The beads were washed with lysis buffer and
resuspended in reducing sample buffer. The boiled samples were separated
on 7% SDS-PAGE and transferred to a PVDF membrane. The membrane
was blocked with 3% BSA in triethanolamine-buffered saline (TBS)
From www.bloodjournal.org by guest on August 11, 2017. For personal use only.
2096
BLOOD, 15 SEPTEMBER 2002 䡠 VOLUME 100, NUMBER 6
SUN et al
overnight at 4°C. After washing, the membrane was incubated for 60
minutes with streptavidin conjugated with horseradish peroxidase, washed,
and detected by enhanced chemiluminescence (ECL) as described above.
Flow cytometric analysis
Nontransfected and transfected CHO cells were harvested, washed, and incubated on ice with mAbs specific for each subunit, the GPIIb-IIIa complex, or
NMIgG at a final concentration of 40 ␮g/mL. After 60 minutes of incubation, the
samples were washed and incubated with a 1:100 dilution of fluorescein
isothiocyanate (FITC)–conjugated goat-anti-mouse IgG (Jackson Lab, West
Grove, PA) for 60 minutes. NMIgM and FITC-conjugated goat-anti-mouse IgM
(Jackson Lab) were used as the negative control and the secondary antibody for
PAC-1 (mIgM). The samples were washed and subjected to flow cytometric
analysis using a FACScan (Becton Dickinson). Selected flow cytometric analysis
was performed in the presence of RGDW peptide (1 mM), using RGEW at the
same concentration as a negative control.
Quantitation of soluble fibrinogen binding to GPIIb-IIIa
on transfected CHO cells
Fibrinogen was labeled with FITC using a protocol provided by Dr Paul F.
Bray (Johns Hopkins University, Baltimore, MD). Briefly, 4 mg fibrinogen
in 0.1 M sodium bicarbonate buffer, pH 9.0, was mixed with 20 ␮g 10%
FITC dispersed on diatomaceous earth (FITC-Celite, Molecular Probes,
Eugene, OR) and incubated for 30 to 60 minutes at 22°C in the dark. The
labeling reaction was stopped by adding 0.1 mL freshly prepared 1 M
ammonium bicarbonate, pH 8.0, for 10 minutes. Labeled fibrinogen was
separated from Celite by centrifugation, and excess FITC was removed by
dialysis against PBS overnight at 4°C in the dark. Fibrinogen concentration
was determined with the BCA protein assay (Pierce). The ratio of FITC to
fibrinogen was determined by comparing A280 to A493. After sterile
filtration, FITC-conjugated fibrinogen was stored at 4°C until use. FITClabeled BSA was prepared for use as a control for background binding.
Nontransfected and transfected CHO cells were washed and incubated at
22°C with FITC-conjugated fibrinogen at a final concentration of 100
␮g/mL in Hanks balanced salt solution (HBSS) containing Ca⫹⫹ and Mg⫹⫹
(1 mM). After incubation for 60 minutes, the samples were then washed,
diluted, and subjected to flow cytometric analysis.
were lysed on the plates with Triton lysis buffer containing sodium
vanadate (1 mM) and scraped into microcentrifuge tubes. Lysates were
incubated on ice for 30 minutes and clarified supernatants were processed
for pp125FAK immunoprecipitation using a rabbit polyclonal antibody
(Santa Cruz Biotechnologies, Santa Cruz, CA), and protein-A Sepharose
(Pharmacia). Precipitates were separated on 7% SDS-PAGE and transferred
to a PVDF membrane. Phosphotyrosine was detected with mAb, PY20.
Results
The long-range disulfide bond between residues Cys5
and Cys435 of GPIIIa is disrupted by Cys5Ala
or by Cys435Ala substitution
The CHO cells transfected with GPIIb and either WT, Ala5GPIIIa,
or Ala435GPIIIa were established. The presence of these mutations
was confirmed by DNA sequence analysis (Figure 1A). Cys5 and
Cys435 have been reported to form a disulfide bridge that brings the
N-terminal region and EGF repeats of GPIIIa into close physical
proximity.15 Substitution of either cysteine 5 or 435 with alanine
would be predicted to result in conformational change, which
should be visible as a shift in the migration of GPIIIa on a
nonreduced SDS-PAGE gel. As shown in Figure 1B, the WT, Ala5,
and Ala435 forms of GPIIIa run with the same mobility under
reducing conditions. However, under nonreducing conditions, both
Ala5GPIIIa and Ala435GPIIIa migrate more slowly than does WT
GPIIIa. These data indicate that the conformation of GPIIIa has
been altered by the alanine 5 or 435 mutations.
Ala5GPIIIa and Ala435GPIIIa are capable of associating
with GPIIb and the integrins are expressed normally
on the cell surface
The ability of Ala435GPIIIa to associate with GPIIb is shown in
Figure 2A. Both AP3 (specific for the GPIIIa subunit) and AP2
Cell adhesion assays
Cell adhesion assays were performed using vital dye-labeled cells as
described previously.36 The 96-well plates (Immunlon 2, Dynatech Labs,
Chantilly, VA) were coated overnight at 4°C with 0.1 mL PBS containing
different concentrations of fibrinogen (2.5-10 ␮g/mL), 10 ␮g/mL AP2, or
1% BSA. The wells were then washed twice with PBS and blocked with 1%
BSA in PBS at 22°C for 60 minutes. Transfected CHO cells were harvested,
washed twice with MEM, and labeled with 2 ␮M calcein AM (Molecular
Probes) at 37°C for 30 minutes. After washing with HBSS, labeled cells
were counted and suspended in ␣-MEM media (serum-free) at a concentration of 1 to 2 ⫻ 106/mL, then added to each well (1-2 ⫻ 105/well) and
incubated for 60 minutes at 37°C. Nonadherent cells were removed by
washing twice with ␣-MEM media; adherent cells in each well were
examined by microscopy and quantified using a microplate fluorescence
reader (CytoFluor II, Perseptive Biosystem, Bedford, MA) at an excitation
wavelength 485 nm and an emission wavelength 530 nm. The fluorescence
intensity of each well was measured before washing as the total cells added
and that after washing as the adherent cells. Quantitative data of cell
adhesion were expressed as the percentage of the total cells added with that
remained in each well after washing. All experiments were performed in
triplicate and repeated at least 3 times. In selected cell adhesion assays, cells
were preincubated with mAbs (40 ␮g/mL) or with RGDW and RGEW
peptides (2 mM) at 22°C for 20 minutes.
Tyrosine phosphorylation of pp125FAK
Suspended cells were seeded onto plastic dishes that had been precoated
with 10 ␮g/mL fibrinogen. After incubation for 30, 60, or 90 minutes at
37°C, plates were washed twice with ice-cold PBS. Then adherent cells
Figure 1. The mutant forms of GPIIIa containing an alanine instead of a cysteine
at amino acid 5 or 435. (A) DNA sequence analysis of Ala5GPIIIa and Ala435GPIIIa
cDNA. The nucleotide sequence was analyzed on both strands using automated
sequencing. As indicated, the nucleotide substitutions of TG with GC (GCT) resulted
in Cys5Ala and Cys435Ala mutations. (B) Western blot analysis of expressed WT
GPIIIa, Ala5GPIIIa, and Ala435GPIIIa. CHO cells (mock) and stable CHO cell lines
expressing WT GPIIb-IIIa (WT), GPIIb-Ala5IIIa (Ala5), or GPIIb-Ala435IIIa (Ala435)
were solubilized in lysis buffer. Soluble lysates were electrophoresed on 7%
SDS-PAGE (left, reduced; right, nonreduced), and subjected to Western blot with
rabbit polyclonal antibodies specific for GPIIIa.
From www.bloodjournal.org by guest on August 11, 2017. For personal use only.
BLOOD, 15 SEPTEMBER 2002 䡠 VOLUME 100, NUMBER 6
CONSTITUTIVELY ACTIVE GPIIb-IIIa COMPLEXES
2097
AP5 and D3,9,27 each of which binds to LIBS determinants on the
activated form of GPIIb-IIIa complex. The binding of AP5 and D3
to CHO cells expressing WT GPIIb-IIIa was low (Figure 3A, left)
as expected. In contrast, the binding of LIBS antibody AP5 to CHO
cells expressing GPIIb-Ala435IIIa (Figure 3A, upper right), and
the binding of LIBS antibody D3 to CHO cells expressing
GPIIb-Ala5IIIa were constitutively high (Figure 3A, lower middle).
AP5 could not report the conformational change elicited by the
Cys5Ala mutation, as its epitope (GPIIIa residues 1-6)9 is proximal
to the amino acid substitution. Similarly, the ability of D3 to bind
the 435AlaGPIIIa variant is lost due to the fact that its epitope is
near residue 422 of GPIIIa.37 However, the binding of non-LIBS
antibody, AP3, which mapped within the residues 348-421 of
GPIIIa, was not affected by Cys435Ala substitution (Figure 2B).
The activation index of other LIBS antibodies is summarized in
Figure 3B. Taken together, these data suggest that the conformation
of both the Cys5Ala and Cys435Ala forms of the GPIIb-IIIa
is altered.
Figure 2. The association of either Ala5GPIIIa or Ala435GPIIIa with GPIIb and
the cell surface expression of GPIIb-Ala5IIIa and GPIIb-Ala435IIIa complexes. (A) Immunoprecipitation analysis of the association of Ala435GPIIIa with
GPIIb. Nontransfected CHO cells (mock) and transfected CHO cells expressing
WT GPIIb-IIIa (WT) or GPIIb-Ala435IIIa (Ala435) were surface-labeled with biotin,
and solubilized in detergent lysis buffer (see “Materials and methods”). The
recombinant cell surface proteins were immunoprecipitated with mAbs specific
either for GPIIIa subunit (AP3, left), or for the GPIIb-IIIa complex (AP2, right). (B)
Flow cytometric analysis of GPIIb-Ala5IIIa and GPIIb-Ala435IIIa expressed on the
cell surface. Nontransfected (left) and transfected CHO cells were stained with
mAbs specific for GPIIIa (AP3, lower), GPIIb-IIIa complex (AP2, upper), or normal
mouse IgG (NMIgG) as negative control at 40 ␮g/mL, and then subjected to flow
cytometric analysis using a FACScan. These 2 antibodies specifically and
positively stained CHO cells expressing WT GPIIb-IIIa (left), GPIIb-Ala5IIIa
(middle), and GPIIb-Ala435IIIa (right).
(specific for the GPIIb-IIIa complex) immunoprecipitated GPIIbAla435GPIIIa together, demonstrating that the formation of an
integrin complex takes place. Essentially the same results were
seen for the Ala5GPIIIa variant (not shown). The expression of
GPIIb-Ala5GPIIIa and GPIIb-Ala435IIIa complexes on the cell
surface was analyzed by flow cytometry using AP2 and AP3
(Figure 2B), and as shown, these 2 antibodies specifically and
positively stained CHO cells with similar mean fluorescence
intensities. Together, these data demonstrate that neither Cys5Ala
nor Cys435Ala mutations affect the ability of GPIIIa to associate
with GPIIb within cells, and that both Ala5GPIIIa and Ala435GPIIIa
are expressed with GPIIb at normal levels on the cell surface. These
cloned CHO cell lines, which express nearly equivalent amounts of
WT GPIIb-IIIa, GPIIb-Ala5IIIa, or GPIIb-Ala435IIIa were used in
the subsequent analyses.
The conformation of the GPIIb-IIIa complex is altered
by the Cys5Ala and Cys435Ala mutations
To examine whether the Cys5Ala or Cys435Ala substitutions affect
the conformation of GPIIb-IIIa on the CHO cell surface, we
performed flow cytometric analysis on transfected CHO cells using
Figure 3. Flow cytometric analysis of the conformational change of the
surface-expressed GPIIb-Ala5IIIa or GPIIb-Ala435IIIa. (A) The binding of LIBS
antibodies AP5 and D3 to the WT GPIIb-IIIa, GPIIb-Ala5IIIa, and GPIIb-Ala435IIIa
complexes. Transfected CHO cells were incubated with AP5 or D3, followed by
FITC-conjugated goat-anti-mouse IgG. Preimmune normal mouse IgG (NMIgG) was
used to establish background binding. (B) Activation index of WT GPIIb-IIIa,
GPIIb-Ala5IIIa, or GPIIb-Ala435IIIa with different LIBS antibodies. LIBS antibody
binding was expressed as an activation index by standardizing the mean fluorescent
intensity (MFI) for each LIBS mAb to that obtained using AP2 whose binding was not
affected by the mutations. Activation index ⫽ LIBS mAb MFI/AP2 MFI for the patient
and for the control separately. Note that the epitope of CRC54 is located within the
first 100 N-terminal residues of GPIIIa,28 and to date no information on the epitope of
7C7 is available.
From www.bloodjournal.org by guest on August 11, 2017. For personal use only.
2098
SUN et al
BLOOD, 15 SEPTEMBER 2002 䡠 VOLUME 100, NUMBER 6
Both GPIIb-Ala5IIIa and GPIIb-Ala435IIIa complexes
constitutively exist in a ligand binding-competent state
To assess the ability of the GPIIb-Ala5IIIa and GPIIb-Ala435IIIa
complexes to bind ligand, we examined the binding of the
ligand-mimetic antibodies Pl-5531 and PAC-129,30 to transfected
CHO cell lines. Although Pl-55 bound poorly to CHO cells
expressing WT GPIIb-IIIa (Figure 4A), it bound avidly to CHO
cells expressing GPIIb-Ala5IIIa or GPIIb-Ala435IIIa complexes.
Similar results were obtained using PAC-1 (Figure 4B, upper).
Binding was specific because it could be completely inhibited by
excess RGDW peptide (Figure 4B, lower). These data demonstrate
that the GPIIb-Ala5IIIa and GPIIb-Ala435IIIa complexes exist in
an activated, ligand binding-competent state.
Both GPIIb-Ala5IIIa and GPIIb-Ala435IIIa complexes exhibit
constitutive fibrinogen binding
To provide further evidence that the GPIIb-Ala5IIIa and GPIIbAla435IIIa complexes exist in an activated state, their ability to
bind soluble fibrinogen was assessed. As shown in Figure 5,
FITC-conjugated fibrinogen bound to GPIIb-Ala435IIIa–transfected CHO cells nearly 2-fold better than it did to CHO cells
expressing WT GPIIb-IIIa. The binding of FITC-conjugated fibrinogen to GPIIb-Ala435IIIa–transfected CHO cells could be blocked
by RGDW peptide (2 mM), but RGEW peptide at the same
Figure 5. Flow cytometric analysis of soluble fibrinogen binding to the cell
surface-expressed GPIIb-Ala435IIIa. Soluble fibrinogen was labeled with FITC as
described in “Materials and methods.” Transfected CHO cells were incubated for 45
minutes at 22°C with either FITC-labeled fibrinogen or FITC-labeled BSA at a
concentration of 100 ␮g/mL in the presence or absence of RGDW peptide (2 mM),
followed by flow cytometric analysis. The left panel showed fibrinogen binding to WT
GPIIb-IIIa on CHO cells, and the right panel showed the binding of soluble
FITC-conjugated fibrinogen to GPIIb-Ala435IIIa on the transfected CHO cells (top).
The bottom panel showed the data of blocking experiments using RGDW peptide.
concentration did not inhibit this binding. The adhesive properties
of the GPIIb-Ala5IIIa and GPIIb-Ala435IIIa complexes were
further examined using immobilized fibrinogen. CHO cells expressing GPIIb-Ala5IIIa or GPIIb-Ala435IIIa complexes bound and
spread on microtiter wells that had been coated with either low (2.5
␮g/mL) or high (10 ␮g/mL) concentration of immobilized fibrinogen (Figure 6A), whereas CHO cells expressing WT GPIIb-IIIa
failed to adhere to immobilized fibrinogen at low concentration
(Figure 6A, top). The quantitative results of the cell adhesion are
shown in Figure 6B. In addition, whereas LIBS antibodies D3,
AP5, and 7G2, significantly increased the adhesion of WT GPIIbIIIa transfectants to low-density immobilized fibrinogen (Figure
6C), they had little additional effect on the already increased
adhesion of CHO cells expressing GPIIb-Ala5IIIa or GPIIbAla435IIIa. Taken together, these data provide evidence that the
Ala5 and Ala435 mutations result in a conformationally altered
high-affinity GPIIb-IIIa complex.
Tyrosine phosphorylation of pp125FAK in
transfected CHO cells
Figure 4. Flow cytometric analysis of the binding of the activation-dependent
fibrinogen-mimetic antibodies to the surface-expressed GPIIb-Ala5IIIa or GPIIbAla435IIIa. (A) The binding of the activation-dependent fibrinogen-mimetic antibody
Pl-55. Flow cytometric analysis was performed as described in “Materials and
methods.” Note the very low binding of Pl-55 to CHO cells expressing WT GPIIb-IIIa
(left) and the high binding to CHO cells expressing either GPIIb-Ala5IIIa or
GPIIb-Ala435IIIa. (B) The binding of the activation-dependent fibrinogen-mimetic
antibody PAC-1. Flow cytometric analysis was performed in the presence of 2.0 mM
RGEW (upper) or RGDW (lower). Normal mouse IgM (NMIgM) and FITC-conjugated
goat-anti-mouse IgM were used as the negative control and the secondary antibody,
respectively.
Ligand binding and clustering of integrins stimulate outside-in
signaling, manifested by responses that include protein tyrosine
phosphorylation and cytoskeletal reorganization. FAK, a 125-kDa
cytoplasmic tyrosine kinase, is a component of focal adhesions and
is a well-established component of integrin signaling pathways.8 To
assess whether the GPIIb-Ala5IIIa and GPIIb-Ala435IIIa complexes are capable of mediating outside-in signaling response on
cell adhesion, the tyrosine phosphorylation state of pp125FAK in
CHO cells expressing WT GPIIb-IIIa, GPIIb-Ala5IIIa, and GPIIbAla435IIIa was compared. As shown in Figure 7, pp125FAK was not
tyrosine-phosphorylated in nontransfected CHO cells after incubation on immobilized fibrinogen. In contrast, GPIIb-Ala5IIIa and
GPIIb-Ala435IIIa, as well as WT GPIIb-IIIa, transfectants exhibited pp125FAK phosphorylation when bound to immobilized fibrinogen, indicating that these active mutant receptors are able to
mediate outside-in signaling response on cell adhesion.
From www.bloodjournal.org by guest on August 11, 2017. For personal use only.
BLOOD, 15 SEPTEMBER 2002 䡠 VOLUME 100, NUMBER 6
CONSTITUTIVELY ACTIVE GPIIb-IIIa COMPLEXES
2099
Figure 7. Tyrosine phosphorylation of pp125FAK.The indicated CHO cells were
seeded onto plastic dishes that had been precoated with 10 ␮g/mL fibrinogen. After
incubation for 30, 60, and 90 minutes at 37°C, plates were washed twice with ice-cold
PBS. Adherent cells were then lysed and subject to pp125FAK immunoprecipitation
and antiphosphotyrosine (upper panel) or anti-pp125FAK (lower panel) Western blot.
Note that there was a small degree of pp125FAK phosphorylation in cells expressing
“activated” integrins. FAK phosphorylation is thought to be downstream, rather than
upstream, of integrin receptor activation, raising the possibility that postreceptor
occupancy events may be facilitated in these cells.
Discussion
Figure 6. Cell adhesion to immobilized fibrinogen. Transfected CHO cells
expressing different forms of GPIIb-IIIa complexes were labeled with calcein-AM at
37°C for 30 minutes. After washing, the cells were allowed to attach at 37°C for 60
minutes to wells coated with the indicated concentrations of fibrinogen, or BSA as the
control. Nonadherent cells were removed by washing; the adherent cells were
examined by phase microscopy and quantified with a fluorescence plate reader. To
calculate the percentage of the bound cells, nonspecific cell adhesion on BSA-coated
wells has been subtracted. (A) Phase contrast microscopic analysis of cell adhesion
to immobilized fibrinogen. The right panel showed that CHO cells expressing either of
GPIIb-IIIa forms bound normally to immobilized fibrinogen coated at high concentration (10 ␮g/mL). The left panel showed that only CHO cells expressing either
GPIIb-Ala5IIIa or GPIIb-Ala435IIIa complex bound wells coated with low concentration of immobilized fibrinogen (2.5 ␮g/mL), and CHO cells expressing WT GPIIb-IIIa
failed to adhere to immobilized fibrinogen at this low concentration. Magnification
⫻ 20. (B) Quantitative analysis of the cell adhesion. CHO cells expressing WT
GPIIb-IIIa, GPIIb-Ala5IIIa, or GPIIb-Ala435IIIa bound to immobilized fibrinogen in a
dose-dependent manner, whereas the nontransfected CHO cells failed to adhere to
immobilized fibrinogen. (C) Cell adhesion to immobilized fibrinogen (2.5 ␮g/mL)
performed in the presence of LIBS antibodies. CHO cells were pretreated with LIBS
antibodies D3, AP5, or 7G2 (40 ␮g/mL). After washing, cell adhesion was performed
as described above.
In this report, we examine the effect of Cys5Ala and Cys435Ala
substitution of GPIIIa on the adhesive properties of the GPIIb-IIIa
complex. We found that (1) both Ala5GPIIIa and Ala435GPIIIa are
capable of associating with GPIIb and are expressed normally on
the surface of transfected CHO cells; (2) both GPIIb-Ala5GPIIIa
and GPIIb-Ala435IIIa exist in an activated conformational state, as
reported by the constitutive binding of LIBS antibodies such as
AP5 or D3, ligand-mimetic antibodies such as PAC-1 and Pl-55,
and soluble fibrinogen; and (3) as a consequence of its activated
state, both GPIIb-Ala5GPIIIa and GPIIb-Ala435IIIa confer to
transfected CHO cells high-affinity ligand-binding properties on
immobilized fibrinogen. Because both Ala5 and Ala435 mutations
in GPIIIa result in similar effects on the adhesive properties of
GPIIb-IIIa, our studies provide functional confirmation for the
presence of a long-range disulfide bond between Cys5-Cys435,15 and
support the notion that this region participates in the conformational changes associated with receptor activation. Additionally,
these data provide a molecular explanation for the previously
reported ability of mild reducing agents to activate the GPIIb-IIIa
complex and promote platelet aggregation.38,39
GPIIIa contains 56 cysteines, 7 of which are located within the
still-to-be-visualized PSI domain, 4 within the ␤A domain, 4 within
the Ig-like “hybrid” domain, 3 within the flexible “linker 2” region
immediately upstream of the first cysteine-rich EGF repeat, 30
within the 4 EGF repeats themselves, and 8 within the ␤-terminal
domain (␤-TD).14 Of these, 32 cysteines can be visualized, and all
of these are paired. Because EGF repeats are exceedingly well
conserved, an additional 7 disulfide bonds located within EGF-1
and EGF-2 (not directly visualized due to likely mobility of this
segment within the crystal) can be assigned with relative confidence, leaving only 10 unaccounted-for cysteines—7 within the
disordered PSI domain, and 3 within linker 2 (residues 435-452).
Cys5 and Cys435 are found within the PSI and linker 2 regions,
respectively, and if paired as proposed,15 would serve to bring into
close apposition these 2 linearly distant regions of GPIIIa. Since
Cys3Ala mutations in either of these residues have similar effects
on complex formation, surface expression, and cell adhesion, our
studies provide functional evidence that a long-range disulfide
bond between Cys5-Cys435 may indeed exist.
In platelet functional assays, it has been previously shown that
treatment of platelets with the reducing agent dithiothreitol induces
From www.bloodjournal.org by guest on August 11, 2017. For personal use only.
2100
BLOOD, 15 SEPTEMBER 2002 䡠 VOLUME 100, NUMBER 6
SUN et al
platelet aggregation, fibrinogen binding, and GPIIb-IIIa conformational changes.38,39 Similar changes have been observed in other
integrins.40 The studies reported here demonstrate that genetic
disruption of Cys5-Cys435 is sufficient to activate the complex.
Interestingly, disruption of other disulfide bonds near or within the
cysteine-rich EGF repeat region of the molecule, including those at
41 also seem to preferentially activate the
Cys23
598 and Cys560,
GPIIb-IIIa complex. Integrin activation as a result of alterations
within the EGF domains does not appear to be restricted to
mutations involving cysteine residues, because Kashiwagi et al22
have shown that Thr562Asn mutation at the boundary of EGF-3
and EGF-4 also results in production of a constitutively active
complex. Taken together, these data suggest the intriguing possibility that the EGF repeats of the molecule may be intimately involved
in the conformational switch that controls receptor activation.
Mutations outside of this region, even to cysteine residues, do not
appear to affect the activation state of the complex, as previously
shown by Wang et al42 for Cys655, which lies within the C-terminal
␤-TD. Structural studies on the effects of perturbations of the EGF
domains on integrin activation are necessary, however, before their
role in controlling the conformation of the complex can be
established with certainty.
In addition to their physiologic role, both GPIIb and GPIIIa are
known to bear a number of clinically important alloantigenic
determinants, such as PlA1, the expression of which is controlled by
a Leu33Pro substitution within the PSI domain at the N-terminus of
GPIIIa. We have previously shown that disruption of the longrange Cys5-Cys435 disulfide bond results in the production of
GPIIIa isoforms that bind some, but not all, anti-PlA1 alloantibodies, suggesting that mutations in this long-range disulfide bond can
alter the conformation of GPIIIa. This substitution also resulted in
the loss of the epitope for certain LIBS mAbs as reported here and
previously,43 consistent with the data that some LIBS antibodies
recognize epitopes within the EGF domains of GPIIIa.6 Because
the Cys5-Cys435 disulfide bridge of GPIIIa connects the PSI domain
to a region immediately amino terminal to the cysteine-rich EGF
repeat region of GPIIIa, and brings the N-terminal region and the
EGF domains of GPIIIa into physical proximity, this disulfide
bridge may have the potential to regulate the shape of the
ligand-binding pocket, and thereby affect the affinity of GPIIb-IIIa
for its ligands. In the crystal structure, the ␤3-subunit “knee” region
is formed from the conjunction of the hybrid domain, EGF domains
1 ⫹ 2, and the PSI domain, and capable of extreme flexibility.
Consistent with this, our studies provide functional confirmation
for the presence of a long-range disulfide bond between Cys5Cys435,15 and support the notion that this loop participates in
conformational changes associated with receptor activation.
GPIIb-IIIa has been reported to contain endogenous thiol
isomerase activity, predicted from the presence of the tetrapeptide
motif, CXXC, in each of the EGF domains of ␤3.17 This motif
comprises the active site in enzymes involved in disulfide exchange
reactions. Thiol isomerase activity within GPIIb-IIIa is inhibited by
the protein-disulfide isomerase (PDI) inhibitor, bacitracin. Blocking PDI with bacitracin inhibits integrin-mediated platelet adhesion
regardless of the affinity state of the integrin. These data suggest
that disulfide exchange takes place during integrin-mediated platelet adhesion18 and altered thiol bonding within the integrin or its
substrates may be locally modified during GPIIb-IIIa activation.
Although all of cysteines within GPIIIa were previously thought to
form disulfide bonds that stabilize the overall 3-dimensional
structure, these studies suggest that the GPIIIa EGF domains may
contain unpaired cysteines that exhibit the properties of a redox site
involved in integrin activation.16 Modification of free cysteines
within GPIIb-IIIa on the platelet surface prevents activationdependent platelet aggregation,16 and activated state of GPIIb-IIIa
is converted to a resting conformation by incubation with a mixture
of sodium nitroprusside (SNP) and reduced glutathione (GSH).16 In
the constitutively active forms of GPIIb-IIIa reported here, free
cysteines have been introduced into GPIIIa, Cys435 for Ala5GPIIIa
and Cys5 for Ala435GPIIIa. One potential mechanism for the
constitutive activation of mutant GPIIb-IIIa might be that these free
cysteines participate directly in disulfide bond rearrangement. This
notion warrants further investigation.
One fundamental function of integrins is ligand binding, which
in many cases is regulated by a process referred to as inside-out
signaling or integrin activation.4,5 The importance of rapid regulated changes in integrin affinity/avidity is easy to appreciate for
GPIIb-IIIa because platelets must interact productively with fibrinogen or VWF following in vascular injury. The significance of
inside-out signaling and, in particular, affinity modulation for ␣v␤3,
which shares a ␤3 subunit in common with GPIIb-IIIa, is less well
understood, though previous studies indicate that ␣v␤3 has the
potential to be regulated at the level of ligand binding.44,45
Applying “gain-of-function” strategies to study the role of ␣v␤3
integrin activation in angiogenesis, tumor invasion, and bone
absorption may be an interesting and important future line of
investigation.
Acknowledgments
We are grateful to Drs Eric Brown, Lisa Jennings, Alexy Mazurov,
Sandy Shattil, Beat Steiner, and Nathalie Valentin for the generous
supply of monoclonal antibodies used in this study.
References
1. Aota S, Yamada KM. Fibronectin and cell adhesion: specificity of integrin-ligand interaction. Adv
Enzymol Relat Areas Mol Biol. 1995;70:1-21.
2. Hynes RO. Integrins: versatility, modulation, and
signaling in cell adhesion. Cell. 1992;69:11-25.
3. Albelda SM, Buck CA. Integrins and other cell
adhesion molecules. FASEB J. 1990;4:28682880.
4. Shattil SJ, Kashiwagi H, Pampori N. Integrin signaling: the platelet paradigm. Blood. 1998;91:
2645-2657.
5. Ginsberg MH, Du X, Plow EF. Inside-out integrin
signalling. Curr Opin Cell Biol. 1992;4:766-771.
6. Du X, Gu M, Weisel JW, et al. Long range propagation of conformational changes in integrin
␣IIb␤3. J Biol Chem. 1993;268:23087-23092.
7. Solowska J, Edelman JM, Albelda SM, Buck CA.
Cytoplasmic and transmembrane domains of integrin ␤1 and ␤3 subunits are functionally interchangeable. J Cell Biol. 1991;114:1079-1088.
8. Aplin AE, Howe A, Alahari SK, Juliano RL. Signal
transduction and signal modulation by cell adhesion receptors: the role of integrins, cadherins,
immunoglobulin-cell adhesion molecules, and
selectins. Pharmacol Rev. 1998;50:197-263.
9. Honda S, Tomiyama Y, Pelletier AJ, et al. Topography of ligand-induced binding sites, including a
novel cation-sensitive epitope (AP5) at the amino
terminus, of the human integrin ␤3 subunit. J Biol
Chem. 1995;270:11947-11954.
10. Haimovich B, Lipfert L, Brugge JS, Shattil SJ. Tyrosine phosphorylation and cytoskeletal reorgani-
zation in platelets are triggered by interaction of
integrin receptors with their immobilized ligands.
J Biol Chem. 1993;268:15868-15877.
11. Kornberg LJ, Earp HS, Turner CE, Prockop C,
Juliano RL. Signal transduction by integrins: increased protein tyrosine phosphorylation caused
by clustering of ␤1 integrins. Proc Natl Acad Sci U
S A. 1991;88:8392-8396.
12. Fitzgerald LA, Steiner B, Rall SC Jr, Lo SS, Phillips DR. Protein sequence of endothelial glycoprotein IIIa derived from a cDNA clone. Identity
with platelet glycoprotein IIIa and similarity to “integrin.” J Biol Chem. 1987;262:3936-3939.
13. Carrell NA, Fitzgerald LA, Steiner B, Erickson HP,
Phillips DR. Structure of human platelet membrane glycoproteins IIb and IIIa as determined by
From www.bloodjournal.org by guest on August 11, 2017. For personal use only.
BLOOD, 15 SEPTEMBER 2002 䡠 VOLUME 100, NUMBER 6
electron microscopy. J Biol Chem. 1985;260:
1743-1749.
14. Xiong JP, Stehle T, Diefenbach B, et al. Crystal
structure of the extracellular segment of integrin
␣V␤3. Science. 2001;294:339-345.
CONSTITUTIVELY ACTIVE GPIIb-IIIa COMPLEXES
constraint regulates integrin signaling. J Biol
Chem. 1996;271:6571-6574.
25. Pidard D, Montgomery RR, Bennett JS, Kunicki
TJ. Interaction of AP-2, a monoclonal antibody
specific for the human platelet glycoprotein IIbIIIa complex, with intact platelets. J Biol Chem.
1983;258:12582-12586.
36.
16. Yan B, Smith JW. A redox site involved in integrin
activation. J Biol Chem. 2000;275:39964-39972.
26. Newman PJ, Allen RW, Kahn RA, Kunicki TJ.
Quantitation of membrane glycoprotein IIIa on
intact human platelets using the monoclonal antibody, AP-3. Blood. 1995;65:227-232.
37.
17. O’Neill S, Robinson A, Deering A, Ryan M,
Fitzgerald DJ, Moran N. The platelet integrin
␣IIb␤3 has an endogenous thiol isomerase activity.
J Biol Chem. 2000;275:36984-36990.
27. Kouns WC, Wall CD, White MM, Fox CF, Jennings LK. A conformation-dependent epitope of
human platelet glycoprotein IIIa. J Biol Chem.
1990;265:20594-20601.
18. Lahav J, Gofer-Dadosh N, Luboshitz J, Hess O,
Shaklai M. Protein disulfide isomerase mediates
integrin-dependent adhesion. FEBS Lett. 2000;
475:89-92.
28. Mazurov AV, Khaspekova SG, Byzova TV, et al.
Stimulation of platelet glycoprotein IIb-IIIa (␣IIb␤3integrin) functional activity by a monoclonal antibody to the N-terminal region of glycoprotein IIIa.
FEBS Lett. 1996;391:84-88.
39.
29. Shattil SJ, Hoxie JA, Cunningham M, Brass LF.
Changes in the platelet membrane glycoprotein
IIb-IIIa complex during platelet activation. J Biol
Chem. 1985;260:11107-11114.
40.
15. Calvete JJ, Henschen A, Gonzalez-Rodriguez J.
Assignment of disulphide bonds in human platelet
GPIIIa. A disulphide pattern for the beta-subunits
of the integrin family. Biochem J. 1991;274:63-71.
19. Bajt ML, Loftus JC, Gawaz MP, Ginsberg MH.
Characterization of a gain of function mutation of
integrin ␣IIb␤3 (platelet glycoprotein IIb-IIIa). J Biol
Chem. 1992;267:22211-22216.
20. Peterson JA, Visentin GP, Newman PJ, Aster RH.
A recombinant soluble form of the integrin alpha
IIb beta 3 (GPIIb-IIIa) assumes an active, ligandbinding conformation and is recognized by GPIIbIIIa-specific monoclonal, allo-, auto-, and drugdependent platelet antibodies. Blood. 1998;92:
2053-2063.
21. Wippler J, Kouns WC, Schlaeger EJ, Kuhn H,
Hadvary P, Steiner B. The integrin ␣IIb-␤3, platelet
glycoprotein IIb-IIIa, can form a functionally active
heterodimer complex without the cysteine-rich
repeats of the ␤3 subunit. J Biol Chem. 1994;269:
8754-8761.
22. Kashiwagi H, Tomiyama Y, Tadokoro S, et al. A
mutation in the extracellular cysteine-rich repeat
region of the ␤3 subunit activates integrins ␣IIb␤3
and ␣v␤3. Blood. 1999;93:2559-2568.
23. Chen P, Melchior C, Brons NH, Schlegel N, Caen
J, Kieffer N. Probing conformational changes in
the I-like domain and the cysteine-rich repeat of
human ␤3 integrins following disulfide bond disruption by cysteine mutations: identification of
cysteine 598 involved in ␣IIb␤3 activation. J Biol
Chem. 2001;276:38628-38635.
24. Hughes PE, Diaz-Gonzalez F, Leong L, et al.
Breaking the integrin hinge. A defined structural
30. Taub R, Gould RJ, Garsky VM, et al. A monoclonal antibody against the platelet fibrinogen receptor contains a sequence that mimics a receptor
recognition domain in fibrinogen. J Biol Chem.
1989;264:259-265.
31. Steiner B, Trzeciak A, Pfenninger G, Kouns WC.
Peptides derived from a sequence within ␤3 integrin bind to platelet ␣IIb␤3 (GPIIb-IIIa) and inhibit
ligand binding. J Biol Chem. 1993;268:68706873.
32. Valentin N, Visentin GP, Newman PJ. Involvement of the cysteine-rich domain of glycoprotein
IIIa in the expression of the human platelet alloantigen, PlA1: evidence for heterogeneity in the
humoral response. Blood. 1995;85:3028-3033.
38.
41.
42.
43.
33. Graham FL, VanderEb AJ: A new technique for
the assay of infectivity of human adenovirus 5
DNA. Virology. 1973;52:456-467.
34. Wilcox DA, Wautier JL, Pidard D, Newman PJ. A
single amino acid substitution flanking the fourth
calcium binding domain of ␣IIb prevents maturation of the ␣IIb␤3 integrin complex. J Biol Chem.
1994;269:4450-4457.
35. Wang R, Newman PJ. Adhesive and signaling
properties of a naturally occurring allele of glycop-
44.
45.
2101
rotein IIIa with an amino acid substitution within
the ligand binding domain—the Pena/Penb platelet alloantigenic epitopes. Blood. 1998;92:32603267.
Wang R, Shattil SJ, Ambruso DR, Newman PJ.
Truncation of the cytoplasmic domain of ␤3 in a
variant form of Glanzmann thrombasthenia abrogates signaling through the integrin ␣IIb␤3 complex. J Clin Invest. 1997;100:2393-2403.
Kouns WC, Newman PJ, Puckett KJ, et al. Further characterization of the loop structure of
platelet glycoprotein IIIa: partial mapping of functionally significant glycoprotein IIIa epitopes.
Blood. 1991;78:3215-3223.
Zucker MB, Masiello NC. Platelet aggregation
caused by dithiothreitol. Thromb Haemost. 1984;
51:119-124.
Kouns WC, Steiner B, Kunicki TJ, et al. Activation
of the fibrinogen binding site on platelets isolated
from a patient with the Strasbourg I variant of
Glanzmann’s thrombasthenia. Blood. 1994;84:
1108-1115.
Davis GE, Camarillo CW. Regulation of integrinmediated myeloid cell adhesion to fibronectin:
influence of disulfide reducing agents, divalent
cations and phorbol ester. J Immunol. 1993;151:
7138-7150.
Ruiz C, Liu CY, Sun QH, et al. A point mutation in
the cysteine-rich domain of glycoprotein (GP) IIIa
results in the expression of a GPIIb-IIIa (␣IIb␤3)
integrin receptor locked in a high-affinity state
and a Glanzmann thrombasthenia-like phenotype. Blood. 2001;98:2432-2441.
Wang R, Peterson J, Aster RH, Newman PJ. Disruption of a long-range disulfide bond between
residues Cys406 and Cys655 in glycoprotein IIIa
does not affect the function of platelet glycoprotein IIb-IIIa. Blood. 1997;90:1718-1719.
Sun Q-H, Liu C-Y, Newman PJ. Disruption of cystine435 of GPIIIa results in the loss of the epitope
for some CD41/CD61 monoclonal antibodies. In
Kishimoto T, eds. Leucocyte Typing VI. New York,
NY: Garland Publishing; 1997:645-647.
Byzova TV, Plow EF. Activation of ␣v␤3 on vascular cells controls recognition of prothrombin.
J Cell Biol. 1998;143:2081-2092.
Bennett JS, Chan C, Vilaire G, Mousa SA, DeGrado WF. Agonist-activated ␣v␤3 on platelets
and lymphocytes binds to the matrix protein osteopontin. J Biol Chem. 1997;272:8137-8140.
From www.bloodjournal.org by guest on August 11, 2017. For personal use only.
2002 100: 2094-2101
doi:10.1182/blood-2002-02-0418 originally published online May
24, 2002
Disruption of the long-range GPIIIa Cys5-Cys435 disulfide bond results in
the production of constitutively active GPIIb-IIIa ( αIIbβ3) integrin
complexes
Qi-Hong Sun, Chao-Yan Liu, Ronggang Wang, Cathy Paddock and Peter J. Newman
Updated information and services can be found at:
http://www.bloodjournal.org/content/100/6/2094.full.html
Articles on similar topics can be found in the following Blood collections
Hemostasis, Thrombosis, and Vascular Biology (2485 articles)
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of
Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.